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Article

Lactic Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen Degradation of Mixed Silage Prepared with Amaranth and Rice Straw

by
Jian Ma
1,†,
Xue Fan
2,†,
Tingting Wu
1,
Jiaxin Zhou
1,
Haozhan Huang
1,
Tianzhen Qiu
1,
Zhewei Xing
1,
Zhihui Zhao
1,
Fuquan Yin
1 and
Shangquan Gan
1,*
1
College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China
2
College of Life Sciences, Northwest Normal University, Lanzhou 730070, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(9), 853; https://doi.org/10.3390/fermentation9090853
Submission received: 25 August 2023 / Revised: 11 September 2023 / Accepted: 12 September 2023 / Published: 19 September 2023
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
The aim of this experiment is to investigate the effects of lactic acid bacteria and cellulase on the fermentation quality, chemical composition, aerobic stability and ruminal degradation characteristics of mixed silage prepared with amaranth and rice straw. Lactic acid bacteria and cellulase were used as silage additives, and the four treatments were as follows: control group (CON, no additive), lactic acid bacteria group (LAB, additive amount was 5 mg/kg fresh matter), cellulase group (CEL, 2 mg/kg) and lactic acid bacteria and cellulase group (LBC, additive amount was the same as in the individual treatments). All treatments were ensiled for 60 days. The dry-matter, crude-protein, neutral-detergent-fiber and acid-detergent-fiber ruminal degradability of silage were analyzed utilizing the nylon bag method. Compared with the CON group, the inoculation of lactic acid bacteria and cellulase individually promoted the fermentation of mixed silage to a certain degree. The combined inoculation of mixed silage significantly increased (p < 0.05) the concentrations of lactic acid and dry matter, while it reduced (p < 0.05) the pH and ammonia nitrogen/total nitrogen, harmful microorganism counts and contents of acetic acid, neutral detergent fiber and acid detergent fiber. In addition, the aerobic stability time of the LBC group was lower (p < 0.05) than that of the other groups. The ruminal degradation rate of dry matter, crude protein, neutral detergent fiber and acid detergent fiber in the LBC group was significantly increased (p < 0.05) compared to the CON group. Overall, the addition of the additives mentioned earlier improved the quality of mixed silage composed of amaranth and rice straw, and the best results were obtained by combining the inoculation of lactic acid bacteria and cellulase.

1. Introduction

Roughage is an important component in the ration of ruminants because of the unique physiological characteristics of gastrointestinal tracts. An adequate provision of high-quality roughage is essential for maintaining the health and production performance of ruminants [1]. Ensiling is an effective strategy to preserve the roughage quality and is commonly used in the diet of ruminants. Corn is one of the main crops for making silage. However, the yield of some crops, such as corn, is scarce in some regions on account of limited land resources, water scarcity and poor soils. Moreover, the crude protein (CP) content and ruminal degradability of corn silage are relatively lower compared to other grass silage (e.g., alfalfa) [2]. The shortage of high-quality roughage has become a critical factor that restricts the stable development of ruminant farming, especially in the dairy cow industry. Thus, taking full advantage of various roughage resources that do not negatively affect the production performance of animals has a significant meaning in dairy farming. Recently, as alternative roughage, the utilization of non-conventional feed resources with high nutrient content and yield has attracted increasing attention.
In ruminant farming, crops that have the ability to adapt to a lack of water, high temperature and poor soils can be utilized as roughage resources under certain harsh conditions [3,4]. As a C4 dicotyledonous crop, amaranth (Amaranthus hypochondriacus) can grow in regions with these severe environments. Forage amaranth has the characteristics of high-yield performance and nutritional value. According to our survey, the yield of amaranth harvested in the heading stage can reach up to 130 t/hm2 (fresh weight) or 20 t/hm2 (dry matter (DM)) [5]. In addition, amaranth has lower contents of oxalic acid, nitrate and lignin compared to other crops such as corn and sorghum [6]. In the budding stage, the CP content of the whole amaranth plant is approximately 14% [5]. Because of this, amaranth has been used as roughage in ruminant production. In dairy cows [7] and fattening lambs [8], partial replacement of corn silage with amaranth silage in the diet has no adverse influence on the health or production performance of animals but reduces the feeding cost.
Silage quality is usually affected by many factors including moisture and the water-soluble carbohydrate (WSC) content of the ensiling materials [9]. Our previous experiment demonstrated that the peaking full-blossom period to the heading period of amaranth was the suitable growth stage for making silage on the basis of fermentation quality and yield [5]. Nevertheless, we found that the moisture content of fresh amaranth is high (approximately 85%) and the WSC is low (approximately 4%). The DM content of agricultural by-product is high, and the yield of agricultural by-product (e.g., rice straw) is abundantly available in China. Therefore, amaranth and rice straw were selected as ensiling materials in the current research. However, although the addition of rice straw to fresh amaranth can reduce the moisture content, it also can dilute the fermentation substrate content and lactic acid bacteria population because rice straw is rich in cellulose and has low epiphytic lactic acid bacteria. Silage additives are beneficial for promoting the fermentation process and enhancing the nutritive value of silage, particularly grass silage. In napier grass silage, the inoculation of lactic acid bacteria can accelerate the fermentation process by utilizing WSC to produce a high concentration of lactic acid and prevent the multiplication of putrefying bacteria [10]. Adding cellulase to rice-straw silage is conducive to degrading structural carbohydrates to provide a fermentation substrate for lactic acid bacteria and then improve the quality of rice-straw silage [11]. On the other hand, the nutrient degradability in the rumen is a key index for evaluating the utilization rate of roughage [12]. Compared with fermentation in vitro, the nylon bag method in situ is more accurate for assessing the rumen degradation rate of forage [13]. In this experiment, we hypothesized that lactic acid bacteria and cellulase may be effective additives that can improve the quality of mixed silage composed of amaranth and rice straw. Therefore, the present research was carried out to investigate the effects of these additives on the chemical components, fermentation characteristics, aerobic stability and nutrient rumen degradability of mixed silage prepared with amaranth and rice straw.

2. Materials and Methods

2.1. Silage Preparation

The amaranth was planted in the test field (longitude 41°25′, latitude 88°40′, altitude 2217 m) with an area of 800 m2 located in Heshuo County, Bayingolin Mongol Autonomous Prefecture, China. The mean annual temperature and precipitation are 11 °C and 59 mm, respectively. Amaranth was planted manually on 10 May 2021 and harvested with reaping hook (Linzhao Industry and Trade Co., Ltd., Linyi, China) in heading period. The whole plant of amaranth was cut to a 5 cm stubble height. Next, the fresh amaranth and rice straw were placed into a forage cutter (Jinhongxing Machinery Co., Ltd., Zhengzhou, China) and chopped into a length of 1.5 to 2 cm. In this experiment, the blending ratio of amaranth and rice stalk was 76%:24% based on the standard that the moisture content of raw materials for ensiling is approximately 65%. The nutritional components of amaranth and rice straw are shown in Supplementary Table S1.

2.2. Experimental Design

In the current experiment, silage additives were commercial lactic acid bacteria (L. plantarum ≥ 1.6 × 1010 CFU/g and L. buchneri ≥ 4.0 × 109 CFU/g, Inner Mongolia Silage Legend Technology Co., Ltd., Hohhot, China) and cellulase (5000 U/g, Xiasheng Biotechnology Co., Ltd., Beijing, China). The amaranth and rice straw were mixed thoroughly, and four groups were as follows: (1) control group with no additive (CON); (2) lactic acid bacteria group (LAB, the additive amount was 5 mg/kg fresh mixed materials); (3) cellulase group (CEL, the additive amount was 2 mg/kg fresh mixed materials) and (4) mixed silage inoculation with lactic acid bacteria and cellulase (LBC, the additive amount was same as in individual groups).
Before ensiling, additives were blended with distilled water and placed into sterile sprayers. Subsequently, the mixtures were evenly splashed on fresh materials layer by layer. Meanwhile, the raw materials in CON group were processed with same amount of distilled water. Next, the mixed materials (approximately 2.2 kg) were compacted and placed into a container (2 L, Hailan Plastic Industry Co., Ltd., Ningbo, China) for fermentation using a wooden stick as a compressor. In this experiment, there were 80 containers in total (4 groups × 4 replicates × 5 time points), and they were stored at room temperature. Samples of ensiling collected at 1, 3, 9, 30 and 60 d were used to analyze the dynamic changes in pH and lactic acid concentration, and samples at 60 d were used for analysis of chemical composition, fermentation profile, aerobic stability and rumen degradation.

2.3. Chemical Composition and Fermentation Quality Analysis of Mixed Silage

After 60 d of fermentation, the fresh silage samples were dried for 48 h (65 °C) to a constant weight. The air-dried silage was smashed with a feed grinder to pass through a 1 mm sieve (Xulang Machinery Equipment Co., Ltd., Guangzhou, China). Then, the samples were analyzed for contents of DM (method 934.01), organic acid (OM, method 942.05) and CP (method 984.13) in accordance with the AOAC procedures [14]. Additionally, the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured with an ANKOM fiber analyzer (ANKOM A2000i, Macedon, NY, USA). The WSC content was determined with the method of sulphuric acid–anthrone colorimetric [15].
To determine the fermentation profile of silage, 20 g fresh samples from each replicate were homogenized in a beaker with 180 mL distilled water and placed into a refrigerator at 4 °C for 24 h. Next, samples were filtered with 4 layers of cheesecloth to collect silage extract. The pH of extract was measured using a pH meter (Jingcheng Instrument Co., Ltd., Qingdao, China). Subsequently, the extract was centrifuged at 10,000× g and 4 °C for 15 min to obtain supernatant. Total nitrogen was determined with a nitrogen analyzer (Kjeltec 8400, FOSS, Gothenburg, Sweden), and ammonia-N (NH3-N) content was analyzed using the method of phenol–hypochlorite reaction [16]. Moreover, the supernatant was used to determine the concentrations of organic acids (lactic acid, acetic acid, propionic acid and butyric acid) using liquid chromatography (EClassical 3200, Dalian, China) according to the procedure of Chen et al. [17].

2.4. Microbial Composition and Aerobic Stability Analysis of Mixed Silage

The number of lactic acid bacteria, mold, aerobic bacteria, yeast and coliform bacteria in silage samples was determined with plate count method in reference to the procedure of Sun et al. [18]. Briefly, 10 g of fresh mixed silage from each replicate was blended with 90 mL sterile water and serially diluted to count the microbial composition in the sterile solution.
After 60 d of fermentation, silage samples were subjected to an aerobic stability test for 5 d according to the method described by de Melo et al. [19]. In this study, the lactic acid bacteria, mold, aerobic bacteria, yeast and coliform bacteria counts were deemed to be spoilage indexes of mixed silage. In addition, aerobic stability time was defined as the number of hours for which the silage maintained stability before reaching 2 °C above environment temperature (24 ± 0.8 °C) after container opening [19]. The temperature of silage was obtained using a digital temperature meter (Anson Intelligent Instrument Co., Ltd., Xian, China).

2.5. Rumen Degradability of Mixed Silage

In this experiment, four Holstein cattle (body weight = 568 ± 15 kg; dry period) fitted with permanent ruminal cannulas were used to analyze DM, CP, NDF and ADF ruminal degradation characteristics of mixed silage using nylon bag method. The cattle were provided with total mixed ration that was designed with reference to the NRC [20]. Feed ingredients and nutritional levels of basal ration are described in Supplementary Table S2. Cows were fed twice daily at 09:00 and 18:00 and had free access to clean water.
The nylon bag method was conducted following the procedure described in our previous study [5]. Briefly, 5 g air-dried silage samples were weighed and sealed into nylon bags (Yaohong Textile Co., Ltd., Suzhou, China). The nylon bag cannot be degraded by rumen microorganisms, and the bag size was 8 × 12 cm with a pore size of 50 μm. Next, the bags were placed into the rumen using the ruminal cannula. The nylon bags were incubated for 4, 8, 16, 24, 36, 48 and 72 h in the rumen of cows. At each time point, the bags had 4 replicates. After taking out the bags from the rumen at different time points, nylon bags were washed with cold tap water until the outlet water was clear. Subsequently, the nylon bags were dried at 65 °C to a constant weight. Samples in bags were weighed and ground by passing through a 1 mm sieve to determine DM, CP, NDF and ADF contents. The nutrient rumen degradability at each time point (t) was calculated with an exponential curve: rumen degradability = a + b (1 − e−ct). The effective degradability was obtained with an equation: effective degradability = a + (b × c)/(c + k) [21]. In these equations, “a” is the rapidly degradable fraction; “b” is the potentially degradable fraction which degrades at a constant fractional rate (c); “e” is the base of natural logarithm and “k” is the ruminal outflow rate. The outflow rate of “k” was 3.1%/h in reference to our previous study [5]. The a, b and c values were obtained using a non-linear regression program in SAS software (version 9.4).

2.6. Statistical Analysis

Before analysis, the normality and homogeneity of data were tested first. Subsequently, all data were analyzed with one-way ANOVA procedure in the SPSS statistical software (version 22.0 for Windows; SPSS, Chicago, IL, USA), with each fermentation container as an experimental unit. Duncan’s test was utilized to evaluate the differences among four treatments. Results are shown as means and standard error of means. Statistical differences are considered significant for p < 0.05 and as a tendency for 0.05 ≤ p < 0.10.

3. Results

3.1. Fermentation Characteristics of Amaranth and Rice-Straw Mixed Silage

Notably, on day 1 of fermentation, the silage pH of the LBC group decreased below 4.2, and that in other groups was above 4.2, among which the pH of the CON group was greater than 5.0 (Figure 1A). The pH of all treatments was below 4.2 on day 9 of fermentation, and the lowest value was approximately 3.9 in the LBC group. The silage pH in all treatments decreased rapidly before fermentation on day 9 and then remained in a stable fluctuation state. As shown in Table 1, after 60 d of fermentation, the pH of the LBC group was significantly reduced (p < 0.05) compared with that of the CON and CEL groups. Additionally, the ratio of NH3-N to total nitrogen in the CON group was higher (p < 0.05) than that in the other groups.
Figure 1B shows the variation tendency of lactic acid in the ensiling process. With the extension of the ensiling time, the lactic acid content of all treatments gradually increased and reached the maximum value on day 30 of fermentation and then began to decrease. The lactic acid content of the LBC group was higher than that of the other groups throughout the fermentation process. After ensiling for 60 d, the LBC treatment showed the highest (p < 0.05) lactic acid concentration, which was 32.12%, 12.54% and 32.63% higher, respectively, than that of the CON, LAB and CEL treatments. On the contrary, the concentrations of acetic and propionic acids in the LBC group were lower (p < 0.05) than those in the CON and CEL groups. There was no butyric acid in the mixed silage inoculated with lactic acid bacteria. Moreover, the lactic acid/acetic acid in the LBC treatment was elevated significantly (p < 0.05) compared with that in the other treatments.

3.2. Chemical Composition of Amaranth and Rice-Straw Mixed Silage

Compared with the CON and CEL groups, the DM content of the LAB and LBC groups was significantly increased (p < 0.05) (Table 2). There was no obvious difference (p > 0.05) in the OM or WSC contents among the four treatments. However, supplementation with cellulase significantly reduced (p < 0.05) the NDF and ADF contents of mixed silage. Furthermore, the CP content in the LBC group tended to be higher (p = 0.068) than that in the CON group.

3.3. Microbial Population of Amaranth and Rice-Straw Mixed Silage

As illustrated in Figure 2A, mixed silage inoculated with lactic acid bacteria significantly increased (p < 0.05) the number of lactic acid bacteria. Conversely, compared with the LAB and LBC groups, the mold (Figure 2B) and aerobic bacteria (Figure 2C) counts in the CON group were observably elevated (p < 0.05). The yeast (Figure 2D) count of the mixed silage was similar (p > 0.05) among all groups. Nevertheless, the coliform bacteria (Figure 2E) count in the CON and CEL groups was higher (p < 0.05) than that in the LBC group.

3.4. Aerobic Stability of Amaranth and Rice-Straw Mixed Silage

After aerobic exposure for 5 d, the mixed silage inoculated with lactic acid bacteria significantly lengthened (p < 0.05) the aerobic stability time of the silage (Figure 3A). The silage in the LAB and LBC treatments displayed significant elevation (p < 0.05) in the lactic acid bacteria count compared to that in the CON and CEL treatments (Figure 3B). The mold count of the LAB, CEL and LBC groups was 32.27%, 18.05% and 42.68% lower, respectively, than that of the CON group (p < 0.05) (Figure 3C). In addition, the aerobic bacteria count of the LBC group was lower (p < 0.05) than that of the other groups (Figure 3D). All additive treatments significantly reduced (p < 0.05) the number of yeast (Figure 3E). Similarly, the coliform bacteria count of the mixed silage in the LAB and LBC groups was lower (p < 0.05) than that in the CON and CEL groups (Figure 3F).

3.5. Ruminal Dry-Matter Degradation Characteristics of Amaranth and Rice-Straw Mixed Silage

The ruminal DM degradation in the LBC treatment was significantly increased (p < 0.05) when compared to the CON treatment from 4 h to 24 h (Table 3). At 72 h, the DM degradation in the LAB and LBC groups was higher (p < 0.05) than that in the CON group. Obviously, the silage DM degradation rate of the four groups was faster before 24 h and then began to slow down. The rapidly degradable fraction in the LBC group was higher (p < 0.05) than that in the CON and CEL groups. Mixed silage inoculated with lactic acid bacteria significantly increased (p < 0.05) the slowly and total degradable fractions. In addition, the effective degradability of DM in the LBC group was observably elevated (p < 0.05) compared with that in the CON and CEL groups.

3.6. Ruminal Crude-Protein Degradation Characteristics of Amaranth and Rice-Straw Mixed Silage

The ruminal CP degradation in the LBC treatment was significantly increased (p < 0.05) compared to that in the CON and LAB treatments at 4, 16, 24 and 36 h (Table 4). Moreover, the CP degradability of the CEL group was higher (p < 0.05) than that of the CON group at 4, 24 and 36 h. At 48 and 72 h, the LBC group displayed the maximum CP degradability, which was higher (p < 0.05) than that of the CON group. Obviously, before 24 h, the ruminal CP degradation rate was faster and then slowed down for the rest of the time. The total degradable fraction and effective degradability of the CEL and LBC groups were higher (p < 0.05) than those of the CON group. There was no significant difference (p > 0.05) in the slowly degradable fraction among the four treatments. Nevertheless, compared with the CON group, the rapidly degradable fraction in the LBC group was slightly increased (p = 0.062).

3.7. Ruminal Neutral-Detergent-Fiber Degradation Characteristics of Amaranth and Rice-Straw Mixed Silage

The NDF degradability of the CEL group was higher (p < 0.05) than that of the CON and LAB groups at 4 h (Table 5). At 16 and 24 h, the CEL and LBC groups showed significantly increased (p < 0.05) NDF degradability compared with the CON and LAB groups. At 72 h, the NDF degradability of all treatments ranged from 47.02% to 53.12%, and the LBC and CEL groups displayed higher values (p < 0.05) compared to the CON group. The rapidly degradable fraction of all groups was similar (p > 0.05). However, the slowly degradable fraction in the LBC group was higher (p < 0.05) than that in the CON group. Additionally, compared with the CON and LAB groups, the total degradable fraction and effective degradability were significantly enhanced (p < 0.05) in the LBC group.

3.8. Ruminal Acid-Detergent-Fiber Degradation Characteristics of Amaranth and Rice-Straw Mixed Silage

There was no significant difference (p > 0.05) in the ADF degradation rate among all groups from 4 to 24 h (Table 6). However, the LBC group displayed a significantly increased (p < 0.05) ADF degradation rate compared with the CON group from 36 to 72 h. Unlike the DM and CP degradation characteristics, the silage ADF degradation rate was slower before 16 h, and from 16 to 48 h, the degradation rate became faster. The rapidly degradable fraction did not show obvious differences (p > 0.05) among all treatments. Nevertheless, the slowly and total degradable fractions of the LBC group were higher (p < 0.05) than those of the CON and LAB groups. Additionally, compared to the CON group, the effective degradability of the LBC group was increased by 37.95% (p < 0.05).

4. Discussion

The chemical components of forage grass are critical factors in determining the silage quality. According to our previous research results, fresh amaranth had high water content (>80%) and low WSC content (<5%) [5]. Increased moisture content can result in clostridium fermentation which produces butyric acid and then decreases the silage quality [22]. The DM content of agricultural by-product such as rice straw is high. Most rice straw is processed with incineration, which leads to serious environmental pollution and increased greenhouse-gas emissions [23]. A mixture of fresh amaranth and rice straw for ensiling may solve the problem that the quality of amaranth silage is low as well as reduce environmental pollution. Apart from that, silage additive is an effective strategy to promote fermentation and enhance the nutritional value of silage. In our study, rice stalk was selected as the mixed material to improve the amaranth silage quality by inoculating lactic acid bacteria and cellulase.
After ensiling for 60 d, the DM content of mixed silage was significantly increased in the lactic acid bacteria treatments. Moreover, the combined addition of lactic acid bacteria and cellulase slightly increased silage CP content. During the ensiling period, as a result of microbial action, silage has a series of biochemical changes which can lead to the loss of nutritional components [24]. In the research of oat silage, inoculation with lactic acid bacteria increased the DM content [25], which is in accordance with our findings. The reason for this result may be associated with the reduction in effluent production by lactobacillus action that includes approximately 8% DM [26]. In addition, supplementation of L. plantarum is beneficial for the homo-fermentation of silage and can inhibit decomposition in organic substances induced by inadequate production of lactic acid [27], which are conducive to decreasing the nutritional loss. WSC is an important fermentation substrate for lactic acid bacteria. During the fermentation of silage, cellulase can accelerate lactic acid bacteria fermentation by decomposing plant fiber to supply carbohydrate substrates [28]. In the current study, the NDF and ADF contents were obviously reduced in the mixed silage inoculated with cellulase. A previous study found that supplementation of cellulase decreased the NDF and ADF contents of alfalfa, wheat-bran and rice-straw mixed silage [29], which is consistent with our study. Overall, the combination of lactic acid bacteria and cellulase in mixed silage displayed the maximum DM and CP contents and the minimum NDF content, indicating that the treatments with the two additives combined were more effective in improving the nutritive value of amaranth and rice-straw mixed silage.
The pH of high-quality silage is below 4.2 [30]. After silage fermentation for 60 d, the pH of all mixed silage was less than 4.2, indicating that the mixed silage was well stored. All silage pH decreased rapidly before 3 d of fermentation, which was related to rapid microbial reproduction that produced a lot of lactic acid and then reduced the silage pH. The combined addition of lactic acid bacteria and cellulase showed the most obvious effect on silage pH, which dropped to approximately 4.1 on the first day of fermentation. After 60 d of ensiling, the pH of the LBC group was lower than that of the CON and CEL groups. Lactic acid bacteria can produce lactic acid by using WSC as a fermentation substrate, thus reducing silage pH. Although the mixed silage of the LAB treatment had enough lactic acid bacteria, the fermentation substrate was insufficient. As a result, the mixed silage in the LAB group could not produce adequate lactic acid to decrease the pH, as seen by the lactic acid yield. The lactic acid content in each group was low in the early stage of fermentation, and then the activity of lactic acid bacteria was enhanced to produce a large amount of lactic acid, which gradually increased to the maximum. The increased lactic acid content began to inhibit the fermentation of lactic acid bacteria, and the lactic acid content began to decrease until it reached a stable level, which was in line with Nicola et al.’s study [31]. The addition of cellulase supplied a fermentation substance for lactic acid bacteria to produce lactic acid and reduce silage pH. Previous research in mixed silage with soybean residue and corn stover reported that the combined inoculation of lactic acid bacteria and cellulase increased lactic acid yield and decreased silage pH [32], which is consistent with our findings.
The growth of destructive bacteria that can produce butyric acid is suppressed by the increased production of lactic acid [30]. Feeding silage that has a high content of butyric acid increases the incidence of metabolic diseases (e.g., ketosis) in dairy cows [33]. In our study, the mixed silage in the LAB and CEL treatments contained butyric acid, indicating that the silage was contaminated by mold. This finding was further verified by the mold count result. In the production process of propionic and butyric acids, some of the metabolic energy is consumed. Additionally, the transformation of lactic acid to butyric acid results in DM loss [34], which has an adverse influence on the dry feed intake of ruminants. Our research results showed that mixed silage supplementation of lactic acid bacteria and cellulase alone or in combination obviously decreased the propionic acid concentration, and combined inoculation had the lowest value. On the other hand, the silage in the CON group displayed the highest acetic acid content, while the LBC group had the lowest value. The reduced lactic acid bacteria number and WSC concentration of the CON mixed silage may have promoted the transformation from homo-fermentation to hero-fermentation, leading to the increased content of acetic acid in the amaranth and rice-straw mixed silage in the CON group.
As an important index to reflect the level of proteolysis in silage, the NH3-N production is generally related to CP degradation induced by enzymes and the microbial population [35]. In the current study, the ratio of NH3-N to total nitrogen in the LAB, CEL and LBC mixed silage was significantly decreased when compared to that of the CON mixed silage, suggesting that the activity of unwanted proteolytic bacteria was effectively restrained in the silage additive treatments. Among all treatments, the LBC treatment showed the minimum value of NH3-N/total nitrogen. After 60 d of ensiling, the reduction in the NH3-N/total nitrogen of the LGC treatment might have been due to the synergistic effect of the different silage additives in inducing nitrification, which converted NH3-N into nitrate nitrogen [36]. In the future, more experiments should focus on the influence of different silage additives on nitrogen conversion during the ensiling process of amaranth. In addition, the acid environment can inhibit the activity of proteolytic enzymes, which is conducive to preventing proteolysis and reducing NH3-N content during the fermentation process [37]. According to our study, the mixed silage in the LBC group displayed the lowest pH value and maintained an optimum acid environment, which was helpful for relieving the CP breakdown of silage. This finding matches the CP results mentioned earlier. To sum up, mixed silage inoculated with lactic acid bacteria or cellulase could improve fermentation quality, and the best results were found in combined inoculation.
In general, the biochemical changes in silage including NH3-N, lactic acid and butyric acid production are strongly linked with the microbial population. Our results showed that mixed silage inoculated with lactic acid bacteria significantly increased the lactic acid bacteria count and decreased the aerobic bacteria and mold counts. In addition, compared with the CON silage, the coliform bacteria count in the LBC silage was significantly reduced. A previous study on mixed silage with whole-plant corn and peanut vines found that the combined inoculation of cellulase and L. plantarum decreased the number of harmful microorganisms including mold, yeast and coliform bacteria [38], which is basically in accordance with our results. The adequate supply of lactic acid bacteria and WSC could explain the reduction in these microbes. During the ensiling process, mixed silage inoculated with lactic acid bacteria and cellulase had the ability to produce adequate lactic acid to decrease the pH and create an acid environment, which then inhibited the reproduction of harmful microorganisms [30]. In the fermentation process of silage, the yeast, mold and coliform bacteria can produce butyric acid by secreting amino acid decarboxylases [39]. Thus, the elevation of these microorganisms counts in the CON and CEL mixed silage resulted in the detection of butyric acid as seen by the fermentation quality results, which had a negative effect on silage quality. Among all the treatments, the combination of lactic acid bacteria and cellulase showed the greatest effect on reducing the unwanted microorganisms in silage.
The activity of aerobic bacteria in silage begins to enhance after the silage is exposed to the air. Then, a mass of heat is released as a result of the metabolism and consumption of nutrients by microorganisms, leading to an elevated pH and the nutritional loss of silage [40]. Therefore, the variation in temperature is a critical index for assessing the aerobic stability of silage. In the current experiment, the aerobic stability time of the LBC silage was highest, indicating that mixed silage inoculated with lactic acid bacteria and cellulase significantly improved aerobic stability. Among the undesirable microorganisms, yeast is regarded as the promoter of silage spoilage and is closely associated with the increased temperature of silage [41]. We speculate that the improvement in the aerobic stability of the LBC silage was related to the decrease in the yeast count. However, the underlying mechanism of action still requires further research. After exposure to air for 5 d, the LBC group showed an increased lactic acid bacteria count and decreased mold and aerobic bacteria counts, which suggests that the lactic acid bacteria in the LBC silage were able to inhibit the reproduction of harmful microorganisms by maintaining the acid environment within a short period. Coliform bacteria can result in metabolic disorders and increase the incidence of diarrhea and inflammation in dairy cows [42]. Although the coliform bacteria count was elevated after aerobic exposure, the coliform bacteria count was reduced in the lactic acid bacteria treatments compared to the CON and CEL silage. Overall, the aerobic stability of amaranth and rice-stalk mixed silage was improved by adding lactic acid bacteria and cellulase.
Apart from the fermentation characteristics and aerobic stability, the nutrient utilization efficiency of ruminants is an important parameter for comprehensively evaluating silage quality. In our experiment, dairy cows were utilized to study the rumen degradation of mixed silage treated with lactic acid bacteria and cellulase. The DM ruminal degradation rate is positively associated with the feed intake of dairy cows [5]. Recent research found that alfalfa silage inoculated with lactic acid bacteria increased DM ruminal degradation [43]. Consistent with this previous study, our results showed that the DM degradability at 72 h and the effective degradability in the rumen of the LBC group were highest, which might have been associated with the elevated DM content in the LBC mixed silage. Likewise, the CP degradation rate in the rumen of the LBC group displayed the maximum value. Previously, a study found that higher CP content in roughage was beneficial for improving ruminal CP degradation [44], which is in line with our findings. The ruminal CP degradability is related to the true protein content of the roughage, and the amino acid composition of CP in roughage can also influence the ruminal CP degradation rate [45]. The lactic acid bacteria treatment may make a positive contribution to improving the amino acid composition of amaranth and rice-stalk mixed silage. On the other hand, the CP effective degradability and total degradable fraction of the CEL and LBC treatments were higher than those of the CON group. A reasonable explanation for this finding is that cellulase treatment increased the soluble true-protein content of mixed silage including in the form of non-ammonia N [46], which was verified by the NH3-N result.
A higher fiber degradation rate in the rumen is conducive to the increased concentration of short-chain fatty acids which can provide energy for ruminants to maintain health and productivity [1]. Our study found that combined inoculation of lactic acid bacteria and cellulase increased the ruminal NDF and ADF degradation rate of amaranth and rice-straw mixed silage. Also, we found that cellulase treatment displayed an obvious improvement in the ruminal NDF degradation rate of mixed silage. The reason might be that the cellulase inoculation of mixed silage promoted the degradation of the connection between polyester and cellulose and then improved the utilization of structural carbohydrates by ruminal microorganisms [47]. In future research, the microbial composition and function of the response of amaranth and rice-straw mixed silage to different additives should be investigated using metagenomics technology. Overall, lactic acid bacteria and cellulase inoculation improved the ruminal degradation of nutrients in amaranth and rice-straw mixed silage, which was beneficial for the utilization of animals.

5. Conclusions

The results of our research provide evidence that lactic acid bacteria and cellulase treatments increase the fermentation quality of mixed silage with amaranth and rice straw to some extent. The combined inoculation of lactic acid bacteria and cellulase increases nutrient contents, promotes fermentation, decreases the counts of harmful microorganisms and improves aerobic stability and nutrient ruminal degradation in mixed silage composed of amaranth and rice straw.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9090853/s1, Table S1: Nutritional components of amaranth and rice straw (DM, %); Table S2: Ingredients and nutrient levels of the experimental diet (DM basis).

Author Contributions

Conceptualization, J.M., X.F. and S.G.; methodology, J.M. and T.W.; software, X.F. and J.Z.; validation, J.M. and S.G.; formal analysis, J.M. and X.F.; investigation, J.M., X.F., T.W., J.Z., H.H., T.Q. and Z.X.; resources, Z.Z. and F.Y.; data curation, J.M., X.F. and S.G.; writing—original draft preparation, J.M. and X.F.; writing—review and editing, J.M., X.F. and S.G.; visualization, X.F.; supervision, S.G.; project administration, J.M. and S.G.; funding acquisition, J.M. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the innovative training program for college students (S202310566025) and program for scientific research start-up funds of Guangdong Ocean University (060302052308).

Institutional Review Board Statement

All procedures involving animal care and management used in this experiment were authorized by the Institutional Animal Care and Use Committee of Guangdong Ocean University (Zhanjiang, Guangdong, China; Approval Code: SYXK-2022-087).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00853 g001
Figure 1. The dynamic changes in pH (A) and lactic acid content (B) of mixed silage in different groups. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. DM, dry matter.
Figure 1. The dynamic changes in pH (A) and lactic acid content (B) of mixed silage in different groups. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. DM, dry matter.
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Fermentation 09 00853 g002
Figure 2. Effects of lactic acid bacteria and cellulase on lactic acid bacteria (A), mold (B), aerobic bacteria (C), yeast (D) and coliform bacteria (E) counts of silage. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. FM, fresh matter. Columns with different small letters mean significant differences (p < 0.05).
Figure 2. Effects of lactic acid bacteria and cellulase on lactic acid bacteria (A), mold (B), aerobic bacteria (C), yeast (D) and coliform bacteria (E) counts of silage. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. FM, fresh matter. Columns with different small letters mean significant differences (p < 0.05).
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Fermentation 09 00853 g003
Figure 3. Effects of lactic acid bacteria and cellulase on aerobic stability of silage. (A) aerobic stability time; (B) lactic acid bacteria; (C) mold; (D) aerobic bacteria; (E) yeast; (F) coliform bacteria. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. FM, fresh matter. Columns with different small letters mean significant differences (p < 0.05).
Figure 3. Effects of lactic acid bacteria and cellulase on aerobic stability of silage. (A) aerobic stability time; (B) lactic acid bacteria; (C) mold; (D) aerobic bacteria; (E) yeast; (F) coliform bacteria. CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. FM, fresh matter. Columns with different small letters mean significant differences (p < 0.05).
Fermentation 09 00853 g003
Table 1. Effects of lactic acid bacteria and cellulase on fermentation quality of silage.
Table 1. Effects of lactic acid bacteria and cellulase on fermentation quality of silage.
ItemsTreatmentsSEMp Value
CONLABCELLBC
pH4.16 a3.92 ab4.13 a3.87 b0.0480.049
NH3-N/total nitrogen4.57 a3.43 b2.97 b2.81 b0.198<0.001
Lactic acid (%, DM)5.23 c6.14 b5.21 c6.91 a0.192<0.001
Acetic acid (%, DM)1.39 a1.08 b1.11 b0.77 c0.064<0.001
Propionic acid (%, DM)0.032 a0.010 bc0.018 b0.007 c0.0030.001
Butyric acid (%, DM)0.005ND0.002ND0.0010.055
Lactic acid/Acetic acid3.79 c5.73 b4.76 bc9.27 a0.582<0.001
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. NH3-N, ammonia nitrogen; DM, dry matter; ND, not detected; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
Table 2. Effects of lactic acid bacteria and cellulase on chemical composition of silage (DM basis, %).
Table 2. Effects of lactic acid bacteria and cellulase on chemical composition of silage (DM basis, %).
ItemsTreatmentsSEMp Value
CONLABCELLBC
DM24.44 b28.89 a25.02 b28.40 a0.539<0.001
OM86.6587.0684.1388.560.7630.231
CP6.787.296.957.570.1190.068
WSC2.181.842.032.270.0920.419
NDF59.64 a57.96 ab54.07 bc53.30 c0.8970.013
ADF41.58 a38.53 ab34.58 c35.61 bc0.8920.007
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. DM, dry matter; OM, organic matter; CP, crude protein; WSC, water-soluble carbohydrate; NDF, neutral detergent fiber; ADF, acid detergent fiber; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
Table 3. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of dry matter in silage.
Table 3. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of dry matter in silage.
ItemsTreatmentsSEMp Value
CONLABCELLBC
Time point (%)
423.95 b28.91 ab27.28 ab31.63 a1.0090.032
831.20 b35.65 ab34.41 b40.94 a1.2120.017
1640.47 c47.70 ab43.56 bc51.78 a1.4020.008
2452.36 b57.52 ab52.55 b60.63 a1.3250.052
3659.1962.2860.8464.880.9430.176
4861.5164.2264.8267.150.9410.212
7263.54 b69.13 a66.44 ab70.89 a1.0310.041
Ruminal degradation parameters
a (%)19.68 c23.46 ab21.47 bc23.97 a0.5520.006
b (%)40.79 b46.47 a42.99 b47.46 a0.8210.002
c (%/h)0.0380.0390.0460.0410.0010.154
a + b (%)60.46 c69.93 a64.46 b71.43 a1.266<0.001
Effective degradability (%)42.02 c49.07 ab47.06 b51.00 a0.956<0.001
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. a, rapidly degradable fraction; b, slowly degradable fraction; a + b, total degradable fraction; c, degradation rate of slowly degradable fraction; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
Table 4. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of crude protein in silage.
Table 4. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of crude protein in silage.
ItemsTreatmentsSEMp-Value
CONLABCELLBC
Time point (%)
434.25 b35.14 b39.77 a40.30 a0.789<0.001
846.3746.9950.6252.200.9650.075
1656.02 b58.14 b59.08 b63.58 a0.9710.020
2463.57 c66.29 bc68.73 ab72.06 a1.0520.011
3666.37 c68.66 bc70.76 ab73.67 a0.8840.007
4870.10 b73.09 ab74.17 ab76.69 a0.8350.023
7273.81 b75.77 ab77.35 ab79.47 a0.7810.047
Ruminal degradation parameters
a (%)35.4537.4439.1140.280.7020.062
b (%)38.0138.4439.8239.980.6070.624
c (%/h)0.0480.0560.0510.0550.0010.088
a + b (%)73.46 b75.87 ab78.92 a80.25 a0.9820.041
Effective degradability (%)58.40 b62.17 ab63.74 a65.86 a0.9480.019
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. a, rapidly degradable fraction; b, slowly degradable fraction; a + b, total degradable fraction; c, degradation rate of slowly degradable fraction; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
Table 5. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of neutral detergent fiber in silage.
Table 5. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of neutral detergent fiber in silage.
ItemsTreatmentsSEMp-Value
CONLABCELLBC
Time point (%)
46.05 c6.84 bc9.77 a8.57 ab0.4970.015
810.55 b12.13 ab14.22 a15.05 a0.6410.033
1615.31 b16.63 b19.58 a20.51 a0.6770.004
2423.87 b26.32 b30.05 a32.07 a0.9570.001
3633.90 b37.02 ab39.81 a38.91 a0.8030.025
4842.28 c45.61 bc48.84 ab50.10 a0.9520.003
7247.02 b49.50 ab51.95 a53.12 a0.7960.013
Ruminal degradation parameters
a (%)3.823.864.084.640.3550.867
b (%)43.74 b45.76 ab47.71 ab49.41 a0.7980.047
c (%/h)0.0390.0410.0450.0440.0010.294
a + b (%)47.55 c49.62 bc51.80 ab54.05 a0.7720.004
Effective degradability (%)28.12 c29.94 bc32.26 ab33.45 a0.6660.005
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. a, rapidly degradable fraction; b, slowly degradable fraction; a + b, total degradable fraction; c, degradation rate of slowly degradable fraction; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
Table 6. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of acid detergent fiber in silage.
Table 6. Effects of lactic acid bacteria and cellulase on ruminal degradability and degradation parameters of acid detergent fiber in silage.
ItemsTreatmentsSEMp Value
CONLABCELLBC
Time point (%)
45.625.485.416.070.4290.960
88.759.088.9210.000.4430.792
1610.9311.5412.1912.990.3960.311
2419.9121.6823.1022.580.4880.087
3627.52 b30.79 ab33.22 a34.08 a0.7920.002
4836.92 b38.51 ab42.73 ab44.18 a1.0200.016
7239.71 b41.55 ab44.96 ab48.19 a1.1490.024
Ruminal degradation parameters
a (%)3.083.143.253.450.2670.971
b (%)37.74 b38.36 b41.69 ab46.30 a1.0960.005
c (%/h)0.0270.0300.0340.0360.0020.372
a + b (%)40.81 b41.50 b44.94 b49.75 a1.029<0.001
Effective degradability (%)20.34 b21.90 ab25.00 ab28.06 a1.0230.017
CON, control group; LAB, mixed silage supplementation with lactic acid bacteria 5 mg/kg; CEL, mixed silage supplementation with cellulase 2 mg/kg; LBC, mixed silage supplementation with lactic acid bacteria 5 mg/kg and cellulase 2 mg/kg. a, rapidly degradable fraction; b, slowly degradable fraction; a + b, total degradable fraction; c, degradation rate of slowly degradable fraction; SEM, standard error of mean. In the same row, values with different superscripts differ significantly (p < 0.05).
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MDPI and ACS Style

Ma, J.; Fan, X.; Wu, T.; Zhou, J.; Huang, H.; Qiu, T.; Xing, Z.; Zhao, Z.; Yin, F.; Gan, S. Lactic Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen Degradation of Mixed Silage Prepared with Amaranth and Rice Straw. Fermentation 2023, 9, 853. https://doi.org/10.3390/fermentation9090853

AMA Style

Ma J, Fan X, Wu T, Zhou J, Huang H, Qiu T, Xing Z, Zhao Z, Yin F, Gan S. Lactic Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen Degradation of Mixed Silage Prepared with Amaranth and Rice Straw. Fermentation. 2023; 9(9):853. https://doi.org/10.3390/fermentation9090853

Chicago/Turabian Style

Ma, Jian, Xue Fan, Tingting Wu, Jiaxin Zhou, Haozhan Huang, Tianzhen Qiu, Zhewei Xing, Zhihui Zhao, Fuquan Yin, and Shangquan Gan. 2023. "Lactic Acid Bacteria and Cellulase Improve the Fermentation Characteristics, Aerobic Stability and Rumen Degradation of Mixed Silage Prepared with Amaranth and Rice Straw" Fermentation 9, no. 9: 853. https://doi.org/10.3390/fermentation9090853

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